Interpretive Summary: Crop improvement through biotechnology requires the introduction of single or multiple genes into plants. However, promoters that drive gene expression often interfere with each other making it impossible to control gene expression in a specific tissue or developmental stage. Identifying insulators to prevent promoter interactions are important for the precise engineering of agronomical traits in crops. We have performed a series of studies and identified at least one insulator from petunia. This insulator can efficiently block interference of other promoters in plants and helps engineer the desired agronomical traits in major crops including fruit species.

Technical Abstract:
While a great deal of research has been dedicated towards the identification and characterization of enhancer-blocking insulators in animal systems, there is a paucity of information concerning these elements in plants. Because there appears to be some overlap in the properties of enhancer-blocking insulators and matrix attachment regions (MARs), we tested the Zea mays ADH1 5’ MAR, tobacco Rb7 3’ MAR and a novel MAR-like transformation booster sequence homologue from Petunia hybrida cultivar V26 (TBSV26) for their ability to impede enhancer-promoter interactions in Arabidopsis thaliana. Stable transgenic lines containing vectors in which one of the three MAR elements or a 4 kb control sequence were interposed between the cauliflower mosaic virus (CaMV) 35S enhancer, and an AGAMOUS second intron (AGI-II)::ß-glucuonidase (GUS) fusion were assayed for GUS activity. We found that the TBSV26 MAR-like element, but not the ADH1 or Rb7 MARs, was able to block interactions between the 35S enhancer and AGI-II without compromising the function of either the enhancer or promoter with elements from which they were not insulated. Furthermore, the enhancer-blocking effect does not appear to be a consequence of the length of the insert, since the 4 kb control fragment, which is almost twice the length of the TBSV26 sequence, was not able to inhibit enhancer-promoter communication.